The present invention relates to a system having hybridization chambers for hybridizing nucleic acid samples, proteins, or tissue sections immobilized on slides. In this case, each hybridization chamber is defined between one of these slides and a cover as an essentially gap-shaped chamber which is essentially fillable with a liquid. Each cover is positioned in relation to a slide in such a way that the hybridization chamber is sealed to the surrounding air. Such a system includes a device for preventing air bubbles in the hybridization chambers. In addition, the present invention relates to a corresponding method for preventing air bubbles in the hybridization chamber of a system for hybridizing nucleic acid samples, proteins, or tissue sections immobilized on slides. According to this method, all essentially gap-shaped hybridization chambers positioned between this slide and a cover are essentially filled with a liquid. In this case, the cover is positioned in relation to the slide so that the hybridization chamber is sealed to the surrounding air.
The use of DNA samples (DNA=deoxyribonucleic acid) and particularly microarrays of such samples provides an important technology to research for simultaneous analysis of thousands of genes. This technology includes the immobilization of DNA samples from many genes on a solid substrate surface, on a glass slide for a light microscope, for example. The DNA samples are preferably positioned in an array of sample spots or “spots”, i.e., in a two-dimensional grid on the substrate and, on the basis of a specific position within such an array, the origin of the corresponding DNA sample may be concluded later. The technology typically includes contacting the DNA sample array with RNA specimen suspensions and/or solutions (RNA=ribonucleic acid) in order to thus detect specific nucleotide sequences in the DNA samples. Typically, specimen suspensions which contain DNA, cDNA, and/or proteins or polypeptides are also used.
RNA specimens may be provided with a so-called “tag” or “label”, i.e., a molecule which emits a fluorescent light having a specific wavelength, for example. Immobilized samples may also include samples containing amino acids (e.g., proteins, peptides) or nucleic acids (e.g., cDNA, RNA). Any arbitrary molecules and/or chemical compounds which hybridize with the immobilized samples or otherwise bond thereto may be included in the specimen added to the immobilized samples.
Under good experimental conditions, the RNA specimens hybridize and/or bond to the immobilized DNA samples and form hybrid DNA-RNA strands together therewith. For each of the immobilized DNA samples and for special RNA specimens, differences in the hybridization among the DNA samples may be determined by measuring the intensity and wavelength dependence of the fluorescence of each individual microarray element and it may thus be found out whether the degree of gene expression varies in the DNA samples assayed. Using DNA microarrays, extensive statements may be made about the expression of large quantities of genes and their expression pattern, although only slight quantities of biological material must be used.
DNA microarrays have been established as successful tools and the devices for performing DNA hybridization are being improved continuously (cf., for example, U.S. Pat. No. 6,238,910 or EP 1 260 265 A1 from the applicant of the present application). These documents disclose a device for providing a hybridization chamber for hybridizing nucleic acid samples on the slide. These devices are implemented as movable in relation to the slide and include an annular seal or sealing surface for sealing the gap-shaped hybridization chamber in relation to the surrounding air, the seal or sealing surface being applied to a surface of this slide. In addition, these devices include lines for supplying and removing media into and from, respectively, the hybridization chamber, as well as a sample supply. An improved temperature control and movement of the liquid having, for example, RNA specimens in relation to the DNA samples immobilized on the slide are also disclosed.
It happens again and again that air bubbles arise when liquids are introduced into the hybridization chamber or even later. However, the attempt has been made (cf., for example, U.S. Pat. No. 6,186,659) to use air bubbles intentionally as an agitation means in order to achieve more thorough mixing of the reagents in the hybridization chamber. In general, however, air bubbles present in the hybridization medium are not desired because they interfere with the liquid film over the immobilized samples, which is usually very thin. This may lead to inhomogeneity of the distribution of reagents in the hybridization medium and therefore to corruption of the hybridization results; in the worst case, larger air bubbles even displace the hybridization medium from parts of the samples immobilized on the slide.
In addition, numerous methods are known from the related art for preventing the spontaneous occurrence of air bubbles or the existence thereof in the chamber. Thus, for example, a non-parallel arrangement of the slide and cover defining the hybridization chamber is suggested (cf. U.S. Pat. No. 5,922,591), or the hybridization media are transported out of the chamber and back in during the entire hybridization process. Admixing agents which reduce the surface tension to the hybridization medium or treating the surfaces of the chamber with water-repellent chemical compounds, with the goal of preventing the formation of air bubbles, is also known.
An arrangement is known from U.S. Pat. No. 6,458,526, using which “bubble halves 140”, made of a gas saturated with solvent, which project into the hybridization chamber, are produced. These “bubble halves” are actually boundary surfaces, shaped like spherical caps, of gas chambers having a defined radius of curvature. These “bubble halves” are located at defined points of the chamber where they may not interfere with the hybridization of the samples. A solvent 160, which is contained in the hybridization medium, is located in a compartment separated from the hybridization chamber. Via this solvent, a saturated atmosphere 150 is maintained, which is constantly connected to the gas chambers behind the “bubble halves 140” (cf. FIG. 2 in U.S. Pat. No. 6,458,526). Therefore, an atmosphere saturated with the solvent is constantly brought to the boundary surfaces shaped like spherical caps and the partial pressure of the solvent present in the hybridization medium is thus influenced so that any air bubbles present shrink and are eliminated. This method has the disadvantage that these boundary surfaces shaped like spherical caps must be provided and maintained using special devices.